Bladder cancer is the 4th most common cancer in men, and the 10th most common in women. In the United States, 72,570 new cases and 15,210 deaths are estimated in 2013. The cost per patient to treat this cancer (diagnosis to death) is ~$150,000, the greatest of any cancer in the medicare population. However, relative to its impact on human health, bladder cancer research is underfunded. Furthermore, little is known regarding the functional determinants of bladder cancer growth. Our lab developed a functional genomewide screen using an shRNA library directed towards the entire human genome to identify suppressors of bladder tumor growth. We identified and validated the glycogen debranching enzyme amylo-alpha-1, 6-glucosidase, 4-alpha- glucanotransferase (AGL) as one such tumor growth suppressor. AGL expression is also inversely associated with prognosis of patients with bladder cancer. While AGL is well studied in the context of glycogen storage disease type III (GSDIII), this is the first instance demonstrating a role for AGL in cancer. Surprisingly, we found that the enzymatic activity of AGL is not necessary for its tumor suppressor function. AGL mutants lacking this activity still suppress proliferation of bladder cancer cells in vitro and in vivo. An important goal is determining how AGL mediates its tumor suppressor function and using this knowledge for therapeutic purposes. Because loss of AGL leads to enhanced tumor growth, genes with expression enhanced by AGL depletion may drive this phenotype. Therefore, we investigated changes in gene expression upon AGL loss in bladder cancer cells. Hyaluronic Acid (HA) Synthase 2 (HAS2) was one of the top upregulated genes in cells with AGL depletion. HAS2 and its product HA have known functions in promoting cancer in several tissues, including bladder. Importantly, depletion of HAS2 blocked tumor growth driven by AGL loss, suggesting HAS2 is a driver of tumor growth upon AGL loss. The first aim of this proposal will address 1) how HAS2 expression is regulated by AGL using HAS2 promoter analysis, 2) if pharmacologically blocking HAS2 activity or the action of HA on human cancer cells in vitro and in vivo can abrogate the growth promoting effect of reduced AGL expression, and 3) finding novel proteins that complex with AGL to elucidate the pathway(s) that lead from AGL to the control of HAS2 expression. Even with the known role of AGL loss in glycogen storage disease, there is no published mouse model with a knockout (KO) of the AGL gene. This model is critically important for the study of the role of AGL in spontaneous bladder carcinogenesis and progression. We have generated mice heterozygous for an AGL KO construct which, upon breeding, should result in such a KO mouse. The second aim of this proposal focuses on characterizing this AGL KO mouse and using it to investigate the impact of AGL loss on the initiation and progression of bladder cancer tumors in mice. Chemical and genetic inducers of bladder cancer will be used in these mice and in wild type littermates. Primary tumor formation and metastasis will be monitored and compared in AGL KOs versus wild type controls.